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Atomic Force Microscope nanolithography onchromosomes to generate single-cell geneticprobesSebastiano Di Bucchianico1*, Anna M Poma1, Maria F Giardi1, Luana Di Leandro1, Francesco Valle2, Fabio Biscarini2

and Dario Botti1

Abstract

Background: Chromosomal dissection provides a direct advance for isolating DNA from cytogeneticallyrecognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that becamevery common in cyto and molecular genetics research and diagnostics. Several reports describing microdissectionmethods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available.Several limitations are imposed by the traditional methods of dissection as the need for a large number ofchromosomes for the production of a probe. In addition, the conventional methods are not suitable for singlechromosome analysis, because of the relatively big size of the microneedles. Consequently new dissectiontechniques are essential for advanced research on chromosomes at the nanoscale level.

Results: We report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of singlechromosomes to generate individual cell specific genetic probes. Besides new methods towards a betternanodissection, this work is focused on the combination of molecular and nanomanipulation techniques whichenable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis ofthe dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can bedirectly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR)and subsequently hybridized to chromosomes and interphasic nuclei.

Conclusions: Atomic force microscope can be easily used to visualize and to manipulate biological material withhigh resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR productsas test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosomenanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to theconstruction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigationof genetic diseases.

BackgroundThe conventional approach to chromosomes microdis-section is based on the use of thin glass needles for thecollection of chromosomes and chromosomal regions.The number of copies of dissected chromosomes neededfor the generation of painting probes, varies from morethan 50 [1] to less than 10 [2]. A modified protocolwhich reduces the copy number of microdissected DNA

fragments has been developed by laser pressure cata-pulting and amplification using linker-adaptor PCR [3].Chromosome recognition is a prerequisite of this techni-que so the chromosome microdissection method waswidely used in genomics research correlated to the G-banding technique.Since its development in 1986 by Binnig et al [4], the

AFM has played a crucial role in the nanoscale biomedi-cal research [5,6]. The AFM is a microscopic systemthat generates a surface topography by using attractiveand repulsive interaction forces between a sharp Si orSiO2 tip attached to a cantilever and a sample. By

* Correspondence: [email protected] of Basic and Applied Biology, University of L’Aquila, Via Vetoio1, L’Aquila 67100, ItalyFull list of author information is available at the end of the article

Di Bucchianico et al. Journal of Nanobiotechnology 2011, 9:27http://www.jnanobiotechnology.com/content/9/1/27

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approaching the cantilever to the sample, the interactionforces can be measured and controlled; upon scanningthe surface it will thus be possible to record the topo-graphy of the sample. This features allow the AFM towork on unstained and uncoated chromosomes [7]. TheAFM imaging reveals that the chromosomes are notuniform in structure but have, along their length, ridgesand grooves that may be related to the G-positive andG-negative bands respectively [8,9]. In this way it is pos-sible to recognize and manipulate chromosomal regionswithout staining and coating.Cytogenetic analysis of MDCC-MSB1, a chicken T-cell

line transformed with Marek’s Disease Virus (MDV), hasbeen performed with both classical methods and AFMdemonstrating a duplication of the short arm of chro-mosome 1, (1p)(p22-p23) [10].It must be underlined that the chicken karyotype con-

sist of 39 chromosomes, 30 of which are classed asmicrochromosomes (MICs) and are cytologically impos-sible to differentiate from each other because of theirsmall size [11]. For this reason it is interesting to usethe AFM as a tool to manipulate chromosomes and togenerate probes for fluorescence in situ hybridization(FISH), confirming the duplication of chromosome 1and making the microchromosomes univocally recogniz-able. The generation of chromosomal painting probesfrom a single unstained chromosome or a single chro-mosomal region can be helpful in studies focusing oncomparative genomics and genomic organization, aswell as in clinical diagnostic of mosaicisms or in hetero-geneous cell populations.Here, we describe the production of specific painting

probes from a single avian microchromosome and a sin-gle chromosomal region using the AFM. When anincreasing force is applied to the microscope tip, ananosize chromosomal region can be dissected away,collecting DNA fragment adherent to the tip. We intro-duce nanolithography on chromosomes surface wherecontiguous line patterns can be generated by a software-controlled pattern generator built in the AFM control-ler. Controlling the lithography software the tip can bemoved with a specified speed along the precise scanninglines. The nanodissected DNA can be amplified throughDOP-PCR [12].

ResultsIn the scanning on the whole metaphase plate the chro-mosome object of nanolitographic dissection has beenidentified. AFM imaging allows the identification of apattern of banding as well as a fibrous structure (withdiameter of around 50 nm). Structural protrusions alongthe chromosome correspond to the “G-positive” bandsthus making the region to be dissected recognizable witha topographical banding [10]. The band (1p)(p22-p23),

that results duplicated in one of the two homologouschromosomes, has been selected in the unduplicatedhomologue to be dissected in order to produce a probefor the FISH. The chromatid band cht del(3)(q2.10) thatresults deleted in both chromosomes has been selectedto be dissected (Figure 1) and the probe generated. Theaim was to show the duplication with molecular methodsand to confirm the ability to identify a single chromatidband with the topographical banding. A microchromo-some has been likewise selected in order to show its uni-vocal recognizability with hybridization molecularmethods, given the non univocal recognizability with tra-ditional cytogenetic methods (Figure 2). Here, we showthat DOP-PCR can be applied to a single unstained chro-mosome or a single chromosomal region without topoi-somerase treatment normally used in the experiments ofchromatin dissection. The results of the DOP PCR per-formed with the nanodissected chromosome 1, the singlenanodissected chromatid of chromosome 3 and the sin-gle microchromosomes nanodissections were examinedin 1% agarose gel electrophoresis and show a bandingpattern between 200 and 600 bp (Figure 3). The templateDNA concentration was comprised from 1 mg/ml and1.5 mg/ml with 260/280 absorbance of 1.7-1.9. Theamplified DNA concentrations were determined byquantitative agarose gel electrophoresis and spectropho-tometric analysis: for all the samples, the concentrationsobtained after DOP PCR were no proportional to the dif-ferent forces applied (5-10 μN) for the dissections, indi-cating that the increase in the depth of the dissection and

Figure 1 Topographic AFM micrograph of chromosomes 3after DNA extraction. Upon localization of the chromosomeregion to be dissected the AFM microscope is switched in ContactNanolithography Mode and the probe is scanned at high force (5-10 μN) several time for few lines (up to 8) perpendicularly to thechromatide. The cross sectional analysis of the cut site reveals a fullwidth at half-maximum height of around 50 nm.


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so in the quantity of the extracted DNA do not affect thequantity of the amplified product.The band specific probe of duplication (1p)(p22-p23)

is generated with Biotin-11-dUTP and applied to inter-phase nuclei (Figure 4). The fluorescent signals were asbright and clear as commercial probes. The probe ofsingle nanodissected chromatid of chromosome 3 ishybridized in interphase nuclei in two distinct spots.In Figure 5 FISH using DOP-PCR products of the

nanodissected microchromosomes is shown. By DOP-PCR of single nanodissected chicken MICs, we havegenerated a chromosome painting probe (Figure 5). We

apply FISH technology as a rapid method for detectionof MICs aneuploidy (Figure 6). The presence of dual sig-nals in the nuclei and the single spot in metaphase isexplained as somatic mosaicism. About 40% of interpha-sic nuclei and/or metaphase scored shows aneuploidy.

Figure 2 Topographic AFM micrograph of microchromosome.Microchromosome before and after (insert) DNA extraction. Thecross sectional analysis of the cut site reveals a full width at half-maximum height of around 40 nm.

Figure 3 DOP PCR results of the nanodissected chromosome.The nanodissected chromosome 1 (lanes 7 and 8), the singlenanodissected chromatid of chromosome 3 (lanes 4, 5, 6) and thesingle microchromosomes nanodissections (lanes 2 and 3) wereexamined in 1% agarose gel electrophoresis and show a bandingpattern between 200 and 600 bp. In lane 1, PCR reaction with noadded DNA and in lane 9 the positive control (1 μg/μL Cot-1 DNA).

Figure 4 FISH using DOP-PCR products. FISH using DOP-PCRproducts of the nanodissected duplication (1p)(p22-p23).Hybridization of the biotinylated probe DNA is detected with FITC-avidin (green signals). Chromosomes are counterstained with DAPI(blue). The tree signals show that the band (1p)(p22-p23) resultsduplicated in one of the two homologous chromosomes.

Figure 5 FISH using DOP-PCR products. FISH using DOP-PCRproducts of the nanodissected microchromosome. Hybridization ofthe biotinylated probe DNA is detected with FITC-avidin (greensignals). Chromosomes are counterstained with DAPI (blue). ByDOP-PCR of single nanodissected chicken microchromosome, wehave generated a chromosome painting probe.


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The level of somatic mosaicism directly contributes tocarcinogenesis by interfering with the normal division ofcells.Conventional fluorescence microscope make it possi-

ble to observe several Kbp DNA probes in metaphaseand it remains impossible to observe probes havinglength shorter than 1 Kbp without several stages of sig-nal amplification. Our probes have a length between 200and 600 bp. The related fluorescent signal in metaphaseis identifiable, with a conventional fluorescence micro-scope, only for MICs characterized by repeatedsequences that allowed repeated hybridization of ourprobes, thus overcoming the resolution limits of conven-tional microscopy. The probes hybridization obtainedfrom chromosome 1 and chromatid regions of chromo-some 3 is confirmed by the fluorescent signal in inter-phasic nuclei where the DNA appears in a more looseform which allows the visualization by mean of conven-tional fluorescence microscopes.

DiscussionThis work shows clearly that DNA can be mechanicallyextracted by the AFM for subsequent use in molecularcytogenetics. To date, various investigators have appliedthe AFM to the dissection of chromosomes at differentregions [13]. We introduce nanolithography on chromo-somes surface. In our laboratory we have reduced the17 μN applied force for the achievement of hybridiza-tion probes used in Iwabuchi’s work and co-authors[14], until 5 μN, minimum value successfully used by

us. The applied forces are comparable to those used byOberringer and co-workers [15]. In addition we haveremarkably reduced the size of the dissected fragmentfrom 1 μm obtained by Yamanaka and co-authors [16]reaching a length of 400 nm.Our experiments have clearly shown that dissected

DNA can subsequently be used as material for PCRamplification and labeling to generate single-cell geneticprobe for FISH analysis. By means of a conventionalfluorescence microscope it is possible to observe DNAprobes in metaphase having a length of several Kbp andit remains impossible to observe probes having lengthinferior to 1 Kbp without several stages of signal ampli-fication. Our probes have a length from 200 and 600 bp.The hybridization obtained by mean of the chromosome1 and chromatid regions of chromosome 3 generatedprobes is confirmed by the fluorescent signal in inter-phasic nuclei where the DNA appears in a more looserform which allows the visualization by mean of conven-tional fluorescence microscopes. As demonstrated byOberringer and co-workers, Scanning Near-field OpticalMicroscope (SNOM) is presently necessary for the opti-cal visualization of probes with dimensions comparableto those obtained by us [15].Moreover, AFM and other new technologies such as

SNOM, may allow in the future more exhaustive exami-nations of metaphase chromosomes and associationsbetween chromosomal aberrations and diseases at ananoscale level. SNOM/AFM, in fact, can simulta-neously obtain topographic and fluorescent images withnanometer-scale resolution. The application of AFM canbe a useful horizons for human cytogenetic studies suchas in cases of recombination at low copy repeats result-ing in tiny deletion/duplication of DNA (Prader-Williand Angelman syndromes or Charcot-Marie-Tooth dis-ease). A further limitation of classical cytogenetic andlargely molecular techniques is the lack of capacity toasses clinically important characteristics of the targetcells. In patient with multiple myeloma, for example,routine FISH assessment may yield normal resultsowing to the low percentage of diseased plasma cells.Thus, a method to generate single-cell genetic probes isneeded in this type of study.Many advantages are attributable to the use of AFM

techniques. These include the high sensibility, the shorttime required for the application and the low quantityof manipulation or chemical treatments that can affectthe structure of chromosomes. Finally it can not beundervalued the low cost of the application in compari-son to traditional techniques. Recent advances in AFMtechnology have improved the resolution using a liquidenvironment. It will be interesting to continue our stu-dies using these new opportunities in conditions closeto chromosome physiological state.

Figure 6 FISH using DOP-PCR products. FISH using DOP-PCRproducts of the nanodissected microchromosome. Hybridization ofthe biotinylated probe DNA is detected with FITC-avidin (greensignals). Chromosomes and nuclei are counterstained with DAPI(blue). We apply FISH technology as a rapid method for detectionof MICs aneuploidy as is clear from the only signal in metaphase.


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ConclusionsThis work demonstrates how it is possible to generategenetic probes for a single specific cell starting from asmall region of chromosome or chromatid dissected byan AFM tip. We have thus achieved a real metaphasechromosome nanolitography. This strategy opens theway for new applications in research and diagnosticcytogenetics, evolutionary studies or physical mappingof the genome. Small amounts of DNA from specificand recognizable sites can be amplified and biotinylatedusing standard molecular biology techniques to behybridized to metaphase plates and interphase nuclei.Implementing this method using scanning near fieldoptical microscopy for fluorescence imaging, can defi-nitely improve the resolution presently limited by opticalmicroscopy thus achieving the study of specific genomicregion labeled with only few dye molecules.

MethodsCell culture and chromosome suspension preparationMDCC-MSB1 cells were cultured in RPMI medium,supplemented with 10% heat-inactivated foetal calfserum (FCS), 100 μg/ml penicillin, 100 μg/ml strepto-mycin at 37°C in 5% CO2. The reagents for cell culturewere purchased from Laboratoires Eurobio (France). Forchromosome suspension preparation during the loga-rithmic growth phase, Colchicine (Sigma, final concen-tration of 0.05 μg/ml) was added to the cultures thatwere then mixed gently and incubated at 37°C for 3 hprior to experiments. The cells were collected, centri-fuged for 10 min at 1200 rpm, and the supernatant dis-carded. The pellet was gently overlain with 10 ml ofphosphate buffer solution (PBS, pH 7.4) three times andsubjected to hypotonic treatment (0.45% sodium citrate)for 10 min before being fixed dropwise in 10 ml coldfreshly made fixative, methanol/acetic acid (3:1). Thechromosome suspension was then stored in fixative at4°C for at least 12 h.

Slide preparation and topographic bandingThe chromosome suspension was centrifuged at 1200rpm for 10 min, the supernatant discarded, and the pel-let was resuspended in cold freshly made fixative againto see it cleaned up. The last pellet was resuspended in0.8/1.0 ml of fixative. Chromosomes were spread on afrosted microscope slide previously washed in fixativediluted in water and put at -20°C in distilled water forat least 1 h. Slides were checked under a phase-contrastmicroscope to ensure that the cell density was correct,and that there were sufficient well-spread, cytoplasm-free mitoses. The slides were finally air-dried. For GTGBanding (Giemsa banding after Trypsin treatment), afteraging for three days, slides were placed in 0.1% trypsinsolution for 20 seconds, rinsed with 70% ethanol,

washed with water and stained in 5% Giemsa’s solutionin pH 6.8 PBS.For topographic banding with the AFM, the slides were

washed in 2 × SSC (0.15 M NaCl, 0.015 M sodium Citrate)for 10 min. Chromosomes were treated with RNase A(Boehringer) stock solution (20 mg/ml) diluted 1:200 in 2× SSC and incubated for 40 min at 37°C. The slides werethen washed in 2 × SSC for 5 min three times, shaking atroom temperature. For protein digestion 10 μl of Pepsin(Sigma, 100 mg/ml) were added to 100 ml of 0.01 M HCl.The slides were incubated in pepsin solution for 5 min at37°C and washed in pH 7.4 PBS buffer for 10 min at roomtemperature. Finally, the slides were dehydrated in an alco-hol series (30-50-70-90-99% of Ethanol) prior to AFManalysis (NT-MDT SMENA on Olympus IX71 InvertedFluorescence Microscope). To identify the TopographicBands, several cross-line profiles through the long axis ofthe chromosomes were measured and compared to theGTG profiles. The ridges of the chromosomes cross-lineprofiles were associated with the GTG+ bands and thegrooves with the GTG- bands.

Atomic Force Microscopy NanolithographyThe nanodissection experiments were carried on by aSmena AFM (NT-MDT, Zelenograd, Russia) operatedboth in intermittent contact and in contact mode. Thecantilever used were NSG10 (NT-MDT, Zelenograd,Russia), with a resonance frequency of 140-390 KHzand a nominal spring constant of 37.6 N/m. The AFMused for these experiments is coupled with an invertedoptical microscope Olympus IX70 (Olympus, Japan) thatallows finding the proper metaphase nuclei and to posi-tion the cantilever on the chromosomes that composeit. Intermittent contact imaging allows identitying all thechromosomes in the chosen metaphase and locating theproper heterochromatin/euchromatin regions, identifiedby topographic banding, to perform the nanodissectionexperiments. Upon localization of the chromosomeregion to be dissected the AFM microscope is switchedin Contact Nanolithography Mode and the probe isscanned at high force (5-10 μN) several times (4 to 6)for few lines (up to 8) perpendicularly to the chroma-tide, this procedure allows the tip to remove the portionof chromatine corresponding to the scanned lines. Thecantilever is then lifted immediatly and stored in therecovery buffer for the further DNA amplification. Toverify the lithographic dissection, imaging is performedon the same chromosome that underwent the procedureto see the effective missing portion.

Degenerate Oligonucleotide-primed Polymerase ChainReaction (DOP-PCR)DOP-PCR of nanodissected chromosomes was per-formed in a MyCycler ™ Thermal Cycler (BIORAD


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Laboratoires, USA). The premixed double concentratedDOP-PCR master mix contains 3 U AccuSure DNAPolymerase (Bioline USA Inc.) in 120 mM Tris-HCl, 60mM (NH4)2SO4, 20 mM KCl, 4 mM MgSO4, pH 8.3,MgCl2 3 mM, Brij 35 (Sigma) 0.02% (v/v), dNTPs 0.4mM. The DOP-PCR reactions was directly performed inthe sterile tubes containing the dissected chromosomefragments adhered to the AFM tip in Tris-HCl 40 mMpH 8.3, MgCl2 20 mM, NaCl 50 mM. In every tubewere added 2 μM 6 MW primer (5’CCGACTC-GAGNNNNNNATGTGG3’, MWG Eurofins Operon,Germany), the DOP-PCR master mix and sterile waterto a final volume of 50 μl. Primary amplification wasperformed with the following cycling parameters: initialdenaturation at 95°C for 5 min, 5 low stringency cyclesof 94°C for 1 min, 30°C for 1.5 min, 3 min transition of30°to 72°C and 72°C for 3 min, followed by 35 highstringency cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min and a final extension of 7 min at 72°C. ThePCR products were analyzed by electrophoretic separa-tion on 1% agarose gel. 5 μl of the primary PCR pro-ducts were labeled with Biotin-11-dUTP ( Fermentas) ina secondary PCR reaction. The 50 μl labelling reactioncontained 1.25 U Taq DNA Polymerase (Fermentas) in10 mM Tris-HCl pH 8.8, 50 mM KCl, 0.08% NonidetP40, 2 mM MgCl2, 0.2 mM dATP, dCTP and dGTP,100 μM dTTP and 80 μM Biotin-11-dUTP (Fermentas).Cycling parameters were: initial denaturation at 94°C for3 min, 20 cycles of 94°C for 1 min, 56°for 1 min, 72°for30 sec and final extension for 3 min. Labeled productswere recovered by ethanol precipitation and 500 ng ofbiotinylated products with 100 fold excess of ChickenCot-1 DNA were resuspended in hybridization solution(50% deionized formamide, 2X SSC, 10% dextransulfate).

Chicken Cot-1 DNA and Probe preparationChicken Cot-1 DNA (not commercially available) is therepetitive sequence of Chicken genomic DNA used as acompetitor to inhibit hybridization of repeats presentwithin DNA probes. Total genomic DNA was isolatedand boiled for 90 min to obtain fragments size of 300-600 bp. The fragmented DNA (1 mg/ml) was denaturedin 0.3 M NaCl at 95°C for 10 min and then allowed toreanneal at 65°C for 6 min. An equal volume of ice-cold2× S1 nuclease buffer and S1 nuclease (Fermentas) wasadded and incubated at 37°C for 30 min. An equalvolume of 25:24:1 phenol:chloroform:isoamyl alcoholwas added and mixed well inverting the tube for 10-15times and then centrifuged for 10 min at 5000 rpm atroom temperature. The supernatant was transferred intoa new tube and a equal volume of chloroform wasadded, mixed well and centrifuged for 10 min at 5000rpm at RT. The supernatant was transferred into a new

tube and 1/12th volume of 3 M NaCl was added, mixedwell, and 2.5 volume of 100% ethanol was then addedand incubated at -20°C overnight. The tube was centri-fuged at 5000 rpm for 30 min and the pellet wash whit70% of ethanol, air-dried and resuspended in distilledwater. The Cot-1 DNA were analyzed by electrophoreticseparation on 1% agarose gel and concentration adjustedto 1 μg/μl. The DOP-PCR labelled probes were dis-solved in 50% formamide, 10% dextran sulphate and 2 ×SSC to a final concentration of 50 ng/μl with a 100 foldexcess of chicken Cot-1 DNA.

Fluorescence in situ hybridization (FISH)The slide-mounted cells were placed for 2 min in adenaturing solution (70% deionized formamide/2 × SSC,pH 7.0) at 70°C in a Coplin jar and then rinsed for 2min in ice-cold 70% ethanol to stop the denaturation.The dehydration was continued by incubating slide for 2min each at room temperature in 80-95-100% ethanol.The slides were finally air-dried. 20 μL of hybridizationsolution is denatured at 75°C for 5 min, applied to slideand covered with a 22-mm2 coverslip. Hybridization wasat 37°C overnight in a moist chamber. Slides werewashed in 50 ml of 50% formamide/2 × SSC at 39°C for15 min, 2 × SSC at 39°C for 15 min, 1 × SSC at roomtemperature for 5 min and allowed to equilibrate 5 minin 4 × SSC at room temperature. 50 μL of biotin detec-tion solution (Avidin-FITC, Vector Laboratories) wasapplied and incubated 45 min in a aluminium foil-wrapped moist chamber at 37°C. The slides weresequentially soak in aluminium foil wrapped Coplin jarscontaining room temperature 4 × SSC, 0.1% Triton X-100/4 × SSC, and 4 × SSC 10 min in each solution. Theslide was counterstained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride). FISH signals were cap-tured by a Zeiss Axioplan 2 fluorescence microscopewith epi-illumination and filter set appropriate for thefluorochrome used.

AcknowledgementsThe work was supported by 2010 RIA grants of University of L’Aquila to A.Poma, D. Botti and EU project BIODOT (Sensing BIOsystems and theirDynamics in fluids with Organic Transistors) supported by the SixthEuropean Research Framework Programme under contract NMP4-CT-2006-032352 at the ISMN-CNR, Bologna.

Author details1Department of Basic and Applied Biology, University of L’Aquila, Via Vetoio1, L’Aquila 67100, Italy. 2Institute for Nanostructured Materials, ConsiglioNazionale delle Ricerche ISMN-CNR, Via P. Gobetti 101, Bologna 40129, Italy.

Authors’ contributionsSDB has made substantial contributions to conception and design,acquisition, collection, analysis, and interpretation of data; has drafted themanuscript. MFG has prepared cells, LDL has performed the DOP-PCRexperiments, FV has made substantial contributions for the use of AtomicForce Microscope. FB supported in the AFM experiments and in the criticalrevision. AP and DB were been involved in drafting and revising the


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manuscript critically for important intellectual content. All authors read andapproved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 12 April 2011 Accepted: 28 June 2011Published: 28 June 2011

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doi:10.1186/1477-3155-9-27Cite this article as: Di Bucchianico et al.: Atomic Force Microscopenanolithography on chromosomes to generate single-cell geneticprobes. Journal of Nanobiotechnology 2011 9:27.

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